Bacterial biofilms are found nearly everywhere in nature and are becoming recognized as the cause of persistent infections. Microbial metabolism and phenotype vary rapidly within a biofilm - this is at least partly responsible for their resistance to antimicrobial agents. Dental caries disease is one of the most prevalent and costly bacterial infections in humans. Caries and associated tooth decay are strongly correlated with a sustained pH decrease induced by oral biofilm (oral bacterial films, i.e. dental plaque) production of organic acids. Detailed knowledge is lacking about these organic acid distributions and the metabolic processes involved. This is due in part to: [i] the complexity of biofilms, which contain temporally evolving spatial concentration gradients for substrate, oxygen, organic acids, etc., and [ii] the inadequacy of current microbiological-metabolism methods which are destructive and typically yield no spatial resolution. The objectives of this two-year R21 project are to adapt, improve and apply unique combined nuclear magnetic resonance and fluorescent confocal (NMR/optical) microscopy instrumentation and techniques for time- and biofilm-depth-resolved metabolism studies of live, cariogenic oral biofilms. A two-step cultivation and measurement procedure will provide reproducible biofilm samples for repeatable depth-resolved metabolism measurements in a relevant (oral) growth environments. One and two-species biofilms will be studied in this fashion. NMR spectroscopic imaging and related methods will be employed to map biofilm metabolism as a function of depth with 10-micron resolution. Specific Aims are to: 1) adapt and improve combined NMR/optical microscopy to study oral biofilm physiology, and 2) employ the new capability to monitor dynamic processes relevant to caries in real time. The resulting biofilm technology and metabolic data will significantly improve our understanding of oral disease processes. The resulting technology will generally be applicable to a variety of mixed-species, medically- relevant biofilms, thus providing key metabolic information for battling bacterial diseases. [unreadable] [unreadable] [unreadable]